The major emphasis of this project will be the study of two purified membrane enzymes. They are (1) pyruvate oxidase, isolated from an E. coli mutant, and (2) C55-isoprenoid alcohol phosphokinase, isolated from S. aureus. Pyruvate oxidase is a water-soluble enzyme which oxidatively decarboxylates pyruvate to yield acetate. This FAD-containing enzyme then reduces a membrane bound lipoprotein. Membrane phospholipids as well as some fatty acids, alkyl sulfates and other lipids result in 15-fold stimulation of activity as well as subunit cooperativity not normally observed. Lipid binding sites will be examined and the role of the membrane as an allosteric effector will be pursued. C55-isoprenoid alcohol phosphokinase is an intrinsic membrane enzyme. It catalyzes the transfer of a phosphate from ATP to ficaprenol, a long-chain alcohol. It is not water-soluble and is one of the most hydrophobic proteins isolated. It is purified using organic solvents. This enzyme has an absolute requirement for lipids for activity. The physical state of the lipid matrix provided by the lipid activator seems to determine the degree of activation. This concept will be pursued using well-characterized, purified lipid activators. Bacitracin forms a specific complex with the membrane lipid, C55-isoprenyl pyrophosphate in the presence of a divalent metal ion. The nature of this complex will be studied in detail using magnetic resonance techniques. The nature of the conformational changes in the E. coli envelope in response to disruption of the energy transduction processes is also being examined using fluorescent and EPR membrane probes.